Biocatalytic approach for the synthesis of enantiopure acebutolol as a β1-selective blocker

Article abstract of DOI:10.1002/chir.22444

Abstract A new chemoenzymatic route is reported to synthesize acebutolol, a selective β₁ adrenergic receptor blocking agent in enantiopure (R and S) forms. The enzymatic kinetic resolution strategy was used to synthesize enantiopure intermediat

Full text of DOI:10.1002/chir.22444

CHIRALITY 27:382–391 (2015)
Biocatalytic Approach for the Synthesis of Enantiopure Acebutolol as a
β -Selective Blocker
LINGA BANOTH, NEERAJ SINGH THAKUR, JAYEETA BHAUMIK, AND UTTAM CHAND BANERJEE*
1
†
†
Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research (NIPER), Punjab, India
ABSTRACT
A new chemoenzymatic route is reported to synthesize acebutolol, a selective β1
adrenergic receptor blocking agent in enantiopure (R and S) forms. The enzymatic kinetic reso-
lution strategy was used to synthesize enantiopure intermediates (R)- and (S)-N-(3-acetyl-4-
(
3-chloro-2-hydroxypropoxy)phenyl)butyramide from the corresponding racemic alcohols. The
results showed that out of eleven commercially available lipase preparations, two enzyme prepa-
rations (Lipase A, Candida antarctica, CLEA [CAL CLEA] and Candida rugosa lipase, 62316
[
CRL 62316]) act in enantioselective manner. Under optimized conditions the enantiomeric
excess of both (R)- and (S)-N-(3-acetyl-4-(3-chloro-2-hydroxypropoxy)phenyl)butyramide were
9.9 and 96.8%, respectively. N-alkylation of both the (R) and (S) intermediates with
9
isopropylamine gave enantiomerically pure (R and S)- acebutolol with a yield 68 and 72%, respec-
tively. This study suggests a high yielding, easy and environmentally green approach to synthe-
size enantiopure acebutolol. Chirality 27:382–391, 2015. © 2015 Wiley Periodicals, Inc.
KEY WORDS: β-blockers; lipase; enantioselectivity; transesterification; enantiopure drug
β-blockers are used in the treatment of several disease
approach has been reported to convert the racemic acebutolol
1
conditions such as hypertension, angina pectoris, cardiac
to the enantiopure form. This shows that only limited
methods are available for the enantiopure synthesis of this
important class of drug molecule. Most of the reagents used
are costly and the yield and enantiomeric excess is poor.
The increase in popularity of biocatalysis-based asymmetric
synthesis and our recent work involving the lipase-based
kinetic resolution method for the synthesis of enciprazine
and bunitrolol led our attention to this problem. We tried to
address this issue by devising a new strategy (strategy 4),
which involved the kinetic resolution with lipase converting
the halohydrin 11 to enantiopure halohydrin, followed by
amination to acebutolol.
2
3
4
arrhythmias, myocardial infarction, migraine, and anxiety
5
disorders. They act as competitive antagonists of catechol-
amines at adrenoceptors. Propranolol is a noncardioselective
β-blocker having membrane-stabilizing properties without
intrinsic sympathomimetic activity. However, acebutolol
(
Ac) is a β -selective-blocking agent having both intrinsic
1
sympathomimetic activity and membrane stabilizing proper-
6
,7
ties. The interaction of drugs and membranes is necessary
8
for the biological effect of the drugs. Moreover, numerous
cardiovascular diseases normally are associated with the
modification of membrane lipid composition and structure.9
Acebutolol is used in the treatment of high blood pressure,
abnormal heart rhythms, and sometimes chest pain.1
Similar to other β-blockers, acebutolol is used as a racemic
mixture, despite the fact that its β-blocking activity is
attributed to the S-enantiomer and its major metabolite
S-diacetolol.18 Moreover, it has been reported that both the
enantiomers of adrenergic β-receptor blockers are not only
different from the pharmacodynamic point of view but their
pharmacokinetic properties are also very different.1
Chemically, acebutolol is an aryloxypropanol amine deriva-
tive bearing one chiral center. Generally, the cardioselectivity
and β-blocking activity of S-enantiomers of aryloxypropanol
amine is 50–500 times more than the R-enantiomers. Several
strategies for the synthesis of acebutolol have been reported
,10
1–17
MATERIALS AND METHODS
Analysis
All the enzymatic reactions were carried out in an incubator (Kuhner,
Switzerland) at 200 rpm. Thin-layer chromatography (TLC) plates were
obtained from Merck (Germany) and silica gel (60–120 mesh) from
(SRL, India) was used in column chromatography. A Finnigan-Mat LCQ
instrument (San Jose, CA) with C-18 hypersil ODS (4.6 mm x 15 mm,
5 m) column was used for liquid chromatography, mass spectrometry
9–21
1
13
(
LC-MS) analysis. H NMR and C NMR spectra were obtained with
1
13
Bruker (Billerica, MA) DPX 400 ( H 400 MHz and C 100 MHz), chem-
ical shifts were expressed in δ units relative to the tetramethylsilane
(
3
TMS) signal as an internal reference in CDCl . IR spectra were re-
corded on Nicolet (Madison, WI) FT-IR impact 400 instruments as neat
for liquid samples or KBr pellets for solid samples. Optical rotation was
measured in a Rudolph, Autopol IV polarimeter. The enantiomeric ex-
cess (ee) was determined by high-performance liquid chromatography
2
2–25
in the literature (Scheme 1).
Only a few reports are available for the enantiopure synthe-
sis of this drug molecule. One of the reported strategies
strategy 1) uses chiral halohydrin or glyceryl acetonide to
convert phenol 3 to enantiopure halohydrin 5. This halohy-
drin is subsequently converted to acebutolol by amination
and hydrolysis. In another strategy (strategies 2, 3) the chiral
epichlorohydrin was used for converting phenol 3 to epoxide
(
*Correspondence to: U.C. Banerjee, Department of Pharmaceutical Technol-
ogy (Biotechnology), National Institute of Pharmaceutical Education and
Research (NIPER), Sector 67, S. A. S. Nagar-160062, Punjab, India. E-mail:
ucbanerjee@niper.ac.in
†
The first two authors contributed equally to this work.
Received for publication 17 November 2014; Accepted 26 February 2015
DOI: 10.1002/chir.22444
2
. This epoxide was then converted to acebutolol by ring open-
ing reaction with amine. Finally, the resolution-based
Published online 15 May 2015 in Wiley Online Library
(wileyonlinelibrary.com).
©
2015 Wiley Periodicals, Inc.

SYNTHESIS OF ENANTIOPURE ACEBUTOLOL
383
Scheme 1. Strategies for the synthesis of acebutolol.
(
HPLC) (Shimadzu, Japan, LC-10AT ’pump, SPD- 10A UV-VIS detector)
(RS)-N-(3-acetyl-4-(oxiran-2-ylmethoxy)phenyl)butyramide (2),
a
1
using a Chiralcel OJ-H column (0.46 mm x 250 mm; 5 μm, Daicel
Chemical Industries, Japan) at 254 nm. The conditions were, mobile
phase: hexane: 2-propanol (4:1); flow rate: 1 mL/min; column tempera-
ture: 25 °C.
yellow liquid (82% yield, 4.5 g);
H
NMR (400 MHz, CDCl ):
3
δ = 1.00 (t, J = 7.4 Hz, 3H), 1.67-1.76 (m, 2H), 2.33 (t, J = 7.28 Hz,
2H), 2.64 (bs, 3H), 2.77 (q, J = 2.64 Hz, 1H), 2.91 (t, J = 4.48 Hz,
1
H), 3.39-3.43 (m, 1H), 3.95 (dd, J = 6.48, 11.2 Hz, 1H), 4.47 (dd,
1
3
J = 2.48 H, 11.24 Hz, 1H), 7.10 (d, 1H), 7.75-7.81 (m, 2H); C NMR
Reagents
(100 MHz, CDCl ): δ 12.6, 18.9, 30.6, 38.3, 43.6, 49.6, 70.1, 113.1,
3
N-(3-acetyl-4-hydroxyphenyl)butyramide, (RS)-epichlorohydrin, (R)-
epichlorohydrin, (S)-epichlorohydrin, and the lipase preparations from
Candida antarctica (CAL L4777), Candida rugosa (CRL 62316), Candida
rugosa (CRL 90860), Candida rugosa L-1754 (CRL L1754), Candida rugosa
121.5, 125.9, 127.9, 132.1, 154.5, 173.1, 199.9; MS (APCI) (m/z)
266.43. (R)-N-(3-acetyl-4-(oxiran-2-ylmethoxy)phenyl)butyramide (2),
2
0
a yellow liquid (81% yield, 1.1 g); 96% ee. [α] -20.4 (c 1.0, MeOH) [α]
D
20
-
20.8 (c 0.013, MeOH), op 98%. (S)- N-(3-acetyl-4-(oxiran-2-
D
(CRL 62316), Aspergillus niger (ANL 62301), and porcine pancreas lipase
ylmethoxy)phenyl)butyramide (2), a yellow liquid (84% yield, 1.2 g);
(PPL) were purchased from Sigma (St. Louis, MO). Immobilized lipase in
0
9
4% ee. [α]2 +19.9 (c 1.0, MeOH).
D
sol-gel-Ak from Pseudomonas cepacia (PCL 62279), immobilized lipozyme
from Mucor miehei (MML 62350), lipase A Candida antarctica (CAL
CLEA) were procured from Fluka (Buchs, Switzerland) and lipase AY
Acetyl chloride (2.1 mL, 20 mmol, 1.5 eq) was added to a stirred so-
lution of (RS)-2 (5.5 g, 20 mmol, 1 eq in 100 mL (DCM: water: 1:1).
The consequential reaction mixture was stirred at 5 °C for 2 h and
on completion of the reaction (TLC), the mixture was extracted with
DCM (50 mL) and washed with water. After organic layer separation,
“Amano”30 (CRL LY amino) was purchased from Amano Chemical
(Elgin, IL). The analytical or commercial grade solvents were procured
from various commercial sources. HPLC-grade solvents were obtained
from Sigma.
Na
2 4
SO was added to it to absorb moisture, then filtered and concen-
trated under vacuum. The remainder was purified by silica column
(
(
60-12 mesh) eluting with ethyl acetate: hexane (15:85) to obtain
RS)-11. The (RS)-11 was then subjected to chiral HPLC analysis
Synthesis of (RS)-N-(3-acetyl-4-(3-chloro-2-hydroxypropoxy)phenyl)
butyramide(11). A previously reported method was used to synthesize
(
RS)-N-(3-acetyl-4-(3-chloro-2-hydroxypropoxy)phenyl)butyramide(RS)-11
using a Chiralcel OJ-H column and the two enantiomers (S)- and
(R)- 11 were eluted after 18.7 min and 19.8 min (hexane:2-propanol:
4:1), respectively, and were present in a ratio of 49:51. Following a
similar procedure, the (R)- and (S)-11 were prepared from (S)- and
(R)-2, respectively.
2
6
with some modifications. Briefly, (RS)-4 (2.3 mL, 20 mmol, 1.5 eq.) was
added to the mixture of 3 (4.4 g, 20 mmol, 1 eq.) and K CO (5.5 g,
0 mmol, 2 eq.) in anhydrous MeCN (100 mL) and the reaction mixture
2
3
2
was heated for 28 h under reflux. The reaction mixture was then cooled,
filtered, and washed with MeCN and the combined organic layer was
concentrated using rotavapor. The residue was purified using silica gel
(RS)-N-(3-acetyl-4-(3-chloro-2-hydroxypropoxy)phenyl)butyramide (11),
1
a white solid (92% yield, 5.7 g); H NMR (400 MHz, CDCl
3
): δ 1.02
(60–120 mesh) column chromatography and eluted with hexane:ethyl
(t, J = 6.76 Hz, 3H) 1.75-1.81 (q, 2H), 2.35 (t, J = 7.32 Hz, 2H), 2.62
acetate (9:1) to afford (RS)- 2.
(s, 3H), 3.33 (bs, 1H), 3.75-3.77 (m, 2H), 4.20-4.24 (m, 3H), 6.97 (d,
Chirality DOI 10.1002/chir

3
84
BANOTH ET AL.
J = 8.92 Hz, 1H), 7.20 (bs, 1H), 7.72-7.733 (m, 1H), 7.81-7.84 (m, 1H);
C NMR (100 MHz, CDCl ): δ 15.6, 20.6, 27.9, 40.5, 48.1, 70.9, 72.5,
3
50 mM) to achieve optimum substrate concentration for the reaction.
Samples were analyzed for conversion and enantioselectivity of the
enzymes used.
1
3
1
3
18.9, 120.7, 121.3, 130.3, 134.4, 154.9, 168.3, 195.2; MS (APCI) (m/z)
02.87.
(
R)-N-(3-acetyl-4-(3-chloro-2-hydroxypropoxy)phenyl)butyramide (11),
Preparative-Scale Transesterification Reaction
The resolution of (RS)-11 was performed by subjecting 50 mL
a white solid, (91% yield, 1.4 g); The product was then subjected to chiral
HPLC analysis using chiral OJ-H column, the two enantiomers were
(
20 mmol substrate, 0.31 g) reaction mixture to resolution by CAL CLEA
and CRL 62316 lipases at 37 and 30 °C using vinyl acetate as acyl donor in
,4-dioxane and toluene, respectively. The reaction mixture was filtered
and the solvent was evaporated in rotavapor and the resulting dried resi-
due was subjected to silica column chromatography using hexane:ethyl
acetate (17:3) as a mobile phase. The isolated yield of (S)-11 was 48%,
eluted at t
peak areas of 3.5 and 96.5%, respectively (93% ee).
S)-N-(3-acetyl-4-(3-chloro-2-hydroxypropoxy)phenyl)butyramide (11),
S R
= 17.9 min and t = 16.9 min (80:20::hexane: 2-propanol) with
1
(
a white solid (94% yield, 1.5 g); The product was then subjected to chiral
HPLC analysis using chiral OJ-H column, the two enantiomers were
eluted at t = 17.9 min and t = 16.9 min (80:20::hexane: 2-propanol) with
S R
0
.15 g with ee 99.7%, (Chiralcel OJ-H) and that of (R)-10 was 46%, 0.16 g
peak areas of 97 and 3%, respectively (94% ee).
with ee 98%, (Chiralcel OJ-H) with CAL CLEA enzyme. It was observed
that with CRL 62316 the isolated yield of (R)-11 was 45%, 0.14 g, with
ee 98% (Chiralcel OJ-H) and that of (S)-10 was 48%, 0.17 g with ee 95%
(Chiralcel OJ-H).
Synthesis of (RS)-1-(2-acetyl-4-butyramidophenoxy)-3-chloropropan-
-yl acetate (10). A well-known method was used to synthesize (RS)-10
with some modifications. Briefly, (RS)-10 was synthesized by treating
RS)-11 (0.16 g, 0.5 mmol, 1 eq) with Ac O (0.05 g, 0.5 mmol, 1 eq) in
the pyridine (0.04 g, 0.5 mmol, 1 eq) at 30 °C in a small RBF with magnetic
stirring. After disappearance of (RS)-11 (TLC, 2 h), ice water (50 mL) was
added to the reaction mixture and pH 3 was adjusted with 3 M HCl. The
final product was extracted with ethyl acetate and brine solution. The
organic layer was then separated and concentrated under vacuum to
afford (RS)-10.
2
26
(
2
2 3
Deacylation of (RS)/(R)/(S)-10. A solution of K CO (0.27 g, 2 mmol)
in deionized water (1 mL) was added to a solution of 10 (0.35 g, 1 mmol)
in methanol (5 mLL) and the resultant reaction mixture was allowed to
stir for 2 h at room temperature (30 °C). After completion, the reaction
mixture was extracted with EtOAc (3 x 15 mL) and water (10 mL).
2 4
The combined organic extracts were dried over Na SO and concen-
trated under vacuum to obtain the crude which was purified by silica
gel column chromatography (100–200 mesh) to obtain the corresponding
alcohol.
RS)-11: a light yellow liquid (97% yield, 0.30 g). (R)-11: a light yellow
liquid, (94% yield, 0.29 g); The product was then subjected to chiral HPLC
analysis using Chiralcel OJ-H column, the two enantiomers were eluted at
38
(RS)-1-(2-acetyl-4-butyramidophenoxy)-3-chloropropan-2-yl acetate (10),
1
a yellow liquid (92% yield, 0.17 g); H NMR (400 MHz, CDCl
3
): δ 2.85-
.87 (m, 1H), 2.93-2.96 (m, 1H), 3.39-3.43 (m, 1H), 4.11-4.15 (dd, 1H),
.36-4.40 (dd, 1H), 7.01-7.06 (m, 2H), 7.51-7.59 (m, 2H); 13C NMR
): δ 44.5, 49.8, 69.3, 102.3, 112.68, 116.2, 121.3, 133.8,
2
4
(
(
100 MHz, CDCl
3
1
34.4, 160.0; MS (APCI) (m/z): 344.92
The (RS)-10 was then subjected to chiral HPLC analysis using a chiral
S R
t = 18.7 min and t = 19.8 min (80:20::hexane: 2-propanol) with peak areas
of 1.0 and 99%, respectively, (98% ee). (S)-11: a light yellow liquid, (97%
yield, 0.30 g); The product was then subjected to chiral HPLC analysis
using Chiralcel OJ-H column, the two enantiomers were eluted at
t = 18.7 min and t = 19.8 min (80:20::hexane: 2-propanol) with peak areas
S R
of 99.2 and 0.8%, respectively (99% ee).
OJ-H column, the two enantiomers were eluted at t
= 54 min (83:17: hexane: 2-propanol) with peak areas of 50.8 and 49.2%,
respectively.
S)-1-(2-acetyl-4-butyramidophenoxy)-3-chloropropan-2-yl acetate (10),
R
= 51 min and
t
S
(
a yellow liquid (93% yield, 0.17 g); The product was then subjected to chi-
ral HPLC analysis using a chiral OJ-H column, the two enantiomers were
eluted at t = 51 min and t = 54 min (83:17::hexane: 2-propanol) with peak
R S
Synthesis of (R)/ (S)- 1. The (R)/(S)-11 (0.31 g, 1.0 mmol) was
treated with isopropylamine (0.081 mL, 1.0 mmol) in methanol
(
10 mL) and Et
3
N (0.14 mL, 1.0 mmol) for 12 h under reflux condi-
areas of 5% and 95%, respectively, (90% ee).
tions. Upon reaction completion, the mixture was diluted with ethyl
3
9
acetate (15 mL) and washed with water. The organic layer was
separated and dried on Na SO and concentrated under vacuum.
Enantioselective transesterification of (RS)-11. In a 5 mL conical
flask a mixture of (RS)-11 (20 mM) in 100 μL [BMIM]PF6, vinyl acetate
5.4 mmol) and toluene (900 μL) were taken. Lipases from different
commercial sources (lipase A, Candida antarctica CLEA, Candida
rugosa 90860, Candida rugosa 62316, Candida rugosa L-1754, Candida
cylindracea, Aspergillus niger, porcine pancreas and AY “Amano”30,
immobilized lipase in sol-gel-Ak from Pseudomonas cepacia, immobilized
lipozyme from Mucor miehei, lipase acrylic resin from Candida antarctica)
were used to carry out the reaction. All the enzyme preparations were indi-
vidually added, flasks were then capped and placed in a shaker at 30 °C
2
4
The residue was purified silica gel (60-12 mesh) column chromatogra-
phy eluted with hexane:ethyl acetate (17:3) to obtain (R)/(S)- 1. (R)-
(
2
7
1
acebutolol (1), a white solid (93% yield, 0.31 g); H NMR (400 MHz,
CDCl
2
(
3
): δ 0.98 (t, J = 7.36, 3H), 1.08-1.10 (m, 6H), 1.65-1.74 (m, 2H),
.29 (t, J = 7.4, 2H), 2.66-2.70 (m, 1H), 2.79-2.87 (m, 2H), 4.03-4.11
1
3
m, 3H), 7.08 (d, J = 8.92, 1H), 7.73-7.79 (m, 2H); C NMR (100 MHz,
CDCl ): δ 12.6, 18.9, 21.1, 21.2, 38.3, 48.5, 49.6, 68.34, 71.6, 113.2, 121.6,
25.9, 127.8, 131.8, 154.8, 173.1, 200.2; MS (APCI) (m/z): 325.44.
3
1
(
(
2
0
S)-acebutolol (1), a white solid (90% yield, 0.30 g); 82% ee. [α]D +15.0
c 1.0, EtOH)
(
200 rpm). After the completion of the reaction, the contents were extracted
using isopropyl alcohol and subjected to HPLC analysis to determine the
conversion and the enantiomeric excess.2
8
Resolution of enzymatically prepared (R)/(S)-11. (R)-N-(4-(3-
chloro-2-hydroxypropoxy)-3-methoxyphenyl)butyramide (11), a white
solid (44% yield, 0.66 g); The product was then subjected to chiral HPLC
analysis using chiral OJ-H column, the two enantiomers were eluted at
t = 18.7 min and t = 19.9 min (80:20::hexane: 2-propanol) with peak areas
Optimization of Transesterification Reaction
Various organic solvents such as diethyl ether, 1,4-dioxane, diisopropyl
ether, DCM, toluene, MeCN, benzene, tert-butyl methyl ether, heptane,
and isooctane were used
of (RS)-11. The optimum time was determined by carrying out the reac-
tion and collecting the samples at various time intervals. Various aspects
of enzymatic acylation such as enantioselectivity, conversion, and green-
ness have been reported to be influenced by the acyl donors. The
enantioselectivity and rate of conversion of enzyme-catalyzed kinetic
resolution was therefore studied using various acyl donors. The reaction
S
R
2
9–33
to find their effect on the transesterification
of 1.7 and 98%, respectively (97% ee). (S)-1-(4-butanoylaminophenoxy)-3-
chloropropan-2-yl acetate (10), a yellow liquid (45.3% yield, 0.31 g); The
product was then subjected to chiral HPLC analysis using the chiral
OJ-H column, the two enantiomers were eluted at t = 42.5 min and
R
3
4
35
tS = 38.7 min (hexane: 2-propanol::4:1) with peak areas of 0.600 and
99.4%, respectively (98.8% ee).
temperature also influences the rate and enantioselectivity of the
RESULTS AND DISCUSSION
transesterification reaction.3
6,37
Various enzyme concentrations (15, 30,
4
5, 60, and 90 mg/mL) were used with a fixed substrate concentration
Biocatalytic routes for the synthesis of enantiopure
acebutolol derivatives are preferred over chemical routes
(20 mM). Substrate concentrations were also varied (10, 20, 30, 40, and
Chirality DOI 10.1002/chir

SYNTHESIS OF ENANTIOPURE ACEBUTOLOL
385
due to the green nature of the procedure. Here,
chemoenzymatic routes to prepare (R) and (S)-acebutolol is
proposed using lipase mediated kinetic resolution (Scheme 2).
Preparation of chemical entities using ionic liquid as reaction
medium and the use of mild biocatalysts such as lipase makes
these routes preferable over the use of harsh chemicals and
solvents.
94% ee and optical rotation [α] +4.28 (c 1.0, CHCl )] were
3
synthesized by the reaction between compound 3 and (S)-
and (R)-4, respectively (Scheme 4). Chiral HPLC and
optical rotation analysis was used to determine the optical
purity of synthesized compounds. Here, the nucleophilic
ring opening of oxirane at the least substituted carbon atom
followed by nucleophilic displacement of chlorine atom
through SN2(concerted) mechanism (Scheme 4) may have
been responsible for the inversion of configuration.2
6,40
Fol-
Synthesis of (RS)-N-(3-acetyl-4-(3-chloro-2-
hydroxypropoxy)phenyl)butyramide (11)
lowing the same mechanism, (R)-11 can be prepared from
(
S)-4.
N-(3-acetyl-4-hydroxyphenyl)butyramide (3) was reacted
with 2-(chloromethyl)oxirane (RS)-4 in the presence of
K CO in MeCN under reflux and afforded N-(3-acetyl-4-
(
R)-11 (91% yield and 96% ee) and (S)-11 (94% yield and
9
5% ee) were afforded by the ring opening of (S)- and (R)-2,
2
3
respectively, using acetyl chloride. In the pyridine, the treat-
(
oxiran-2-ylmethoxy)phenyl)butyramide (RS)-2 with 82%
ment of (RS)-11 with Ac O at 4 °C gave the (RS)-10 in 92%
yield. Further, the desired substrate for the lipase catalyzed
kinetic resolution, (RS)-11 (92% yield) was obtained by the
reaction of (RS)-2 with acetyl chloride in DCM and water
2
yield. Using similar procedure acetylation of (R)-11 and (S)-
1
1 afforded, (R)-10 and (S)-10 (91% yield and 94% ee and
9
3% yield and 93% ee, respectively) (Scheme 5).
(
Scheme 3).
Synthesis of Standard Compounds Through the
Screening of Lipases for Kinetic Resolution of (RS)-11
Chemical Route
The substrates (RS)-11 and the standard samples of (R)/
The standard samples of (R)- or (S)-11 and their O-
(S)-11 and (R)/(S)-10 were used to determine the efficient
method for the enzymatic kinetic resolution. Commercial
lipases from different sources were screened using vinyl
acetate as acyl donor for the transesterification of (RS)-11
in toluene (Scheme 6).
acetylated derivatives (R)- or (S)-10 were synthesized to
optimize the enzymatic kinetic resolution conditions. For
this purpose, first (R)-2 [81% yield, 92% ee and optical
rotation [α] +4.22 (c 1.0, CHCl )] and (S)-2 [84% yield,
3
Scheme 2. Retrosynthetic pathway towards the synthesis of enantiopure acebutolol.
Scheme 3. Synthesis of (RS)-11.
Chirality DOI 10.1002/chir

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86
BANOTH ET AL.
Scheme 4. Mechanism of formation of (S)-11 from (R)-4.
Scheme 5. Synthesis of (RS)/(R)/(S)-10.
Scheme 6. Enzymatic kinetic resolution of (RS)-11.
Among the various enzymes used, only two of them (CAL
CLEA and CRL 62316) were found to be better in terms of
conversion and enantioselectivity. For the conversion of
solvents (log P < 2) as compared to apolar solvents (log P > 4).
(RS)-11 is not soluble in vinyl acetate. A detailed solubility
study of (RS)-11 was carried out using eleven various organic
solvents to screen and optimize resolution conditions (Supple-
mentary Material Table 1). It was found that (RS)-11 is insolu-
ble in most of the organic solvents except 1,4-dioxane,
acetonitrile, and DCM; however, in order to screen various
other organic solvents to carry out the lipase catalyzed reac-
tions in interphase, it is mandatory to make (RS)-11 soluble
in some solvents. Further ionic liquids were used to make
(RS)-11 soluble in the presence of various organic solvents
and its miscibility with isopropyl alcohol were tested out. Mis-
cibility of ionic liquids is important for extraction of 11 from
the reaction mixture using isopropyl alcohol as the extracting
solvent (Supplementary Material Table 2). It was found that
(RS)-11 is soluble in [BMIM]BF and [BMIM]PF but insolu-
(
RS)-11 to (R)-11 and (S)-10 the CAL CLEA showed better
activity and the conversion of (RS)-11 to (S)-11 and (R)-10
was most efficiently done by CRL 62316. It is to be noted
that the two enzymes showed opposite enantioselectivity
(
Table 1).
Screening of Organic Solvents
The solvent effect on the enantioselectivity of enzymatic
2
9–32
reactions are well known.
Lipases are maximally used by
the chemists because of their high stability in organic
solvents.33 In this study a number of solvents with different
log P values were investigated for the resolution of (RS)- 11
4
2
(
Table 2). It has been reported in the literature they demon-
4
6
strate that the rates of biocatalytic reactions are less with polar
ble in [EMIM]BF4. Also, it was found that [BMIM]PF was
6
Chirality DOI 10.1002/chir

SYNTHESIS OF ENANTIOPURE ACEBUTOLOL
387
TABLE 1. Lipase-catalyzed transesterification of (RS)-11 with
a
vinyl acetate
Time
(h)
C c
(%)
ee d
(%)
s
eepe
(%)
Configuration
b
f
Type of lipase
E
of 10
CRL AMANO
CAL
CCL
CAL CLEA
CRL 62316
CRL L1754
48
48
48
48
48
48
17
44
26
15
35
21
71
44
59
6.8
3.5
4.7
R
S
R
S
R
R
48.7 89.3 94.1 99.6
48
27
85
18
91
49
61
3.4
a
Conditions: (RS)-11 (20 mM) in toluene (1 mL) was treated with of vinyl ac-
etate (5.4 mmol) at 30 °C in the presence of the enzyme (15 mg/mL).
b
CRL AMANO, CAL, CCL CAL CLEA, CRL 62316, & CRL L1754 lipase,
c
Conversions were calculated from the enantiomeric excess (ee) of (R)-11
(
substrate S) and (S)-10 (product P) using the formula: Conversion (C)
ee /(ee + ee ).
S S P
Enantiomeric excess of (R)/(S)-11 was determined by HPLC analysis
Daicel Chiralcel OD-H column) 80:20; hexane: 2-propanol, 1 mL/min flow
Fig. 1. Course of CAL CLEA catalyzed transesterification of (RS)-11 in 1,4-
dioxane.
=
d
(
rate at 254 nm.
solvent, it was found that mmaximum conversion (C = 49%),
enantiomeric excess of substrate (96%), and enantiomeric
excess of the product (98%) were achieved at 30 h. The enan-
tiomeric ratio and enantiomeric excess of the product
improved up to 30 h (E = 381) (Fig. 1). In toluene, the conver-
sion and enantiomeric excess of substrate increased with
the reaction time. Maximum conversion (49%), enantiomeric
excess of substrate (88%) and product (91%) were achieved
after 30 h of reaction. The enantiomeric ratio increased up
to 30 h (E = 60) (Fig. 2). Slower reacting enantiomers have
the disadvantage of being converted to the undesired isomer
if the reaction time is prolonged, resulting in a less satisfac-
tory enantiomeric excess. Thus, both solvents (toluene and
1,4-dioxane) were chosen and the optimum reaction time
e
Enantiomeric excess of (R)/(S)-10 was determined by HPLC analysis
(
Daicel Chiralcel OD-H column) 80:20; hexane: 2-propanol, 1 mL/min flow
rate at 254 nm.
f
p s p s
E values were calculated using the formula: E = ln [(ee (1 – ee )/(ee + ee )]/
28,41
p P s
ln[(ee (1 + ees)/(ee + ee )].
immiscible with isopropyl alcohol which is important for the
extraction of (R)/(S)-11 and (R)/(S)-10 from the reaction mix-
ture. The solvent effect of CAL CLEA and CRL 62316 for the
kinetic resolution of (RS)-11 was studied using vinyl acetate
as the acyl donor at 30°C. The 1,4-dioxane for CAL CLEA and
toluene for CRL 62316 enzyme preparations were suggested
for the maximum enantioselectivity and enantiomeric excess
of substrate and product as compared to the other solvents.
(
30 h) was used to carry out further studies.
Effect of Reaction Time
Effect of Acyl Donors
CAL CLEA and CRL 62316 catalyzed transesterification
reaction of (RS)-11 were carried out separately in 1,4-dioxane
and toluene, respectively. The chiral HPLC was used to deter-
mine conversion and enantiomeric excess. In the 1,4-dioxane
The enantioselectivity and rate of conversion of CAL CLEA
and CRL 62316-catalyzed kinetic resolution of (RS)-11 was
studied in toluene and 1,4-dioxane, respectively, using
a
TABLE 2. Effect of organic solvent on the enantioselectivity in the resolution of (RS)-11 with lipase .
S-selectivity with CAL CLEA R-selectivity with CRL 62316
b
c
d
e
b
c
d
e
Solvent
Log P
C (%)
ee
s
(%)
ee
p
(%)
E
C (%)
ee
s
(%)
ee
p
(%)
E
Acetonitrile
-0.33
-1.1
1.35
0.85
1.25
2
4
2.5
3.41
3.5
4.5
55.2
49.6
81
87
77
78
84
48.8
77
72
99.9
93.8
82
53
26
80
66
90.4
71
36
80.9
95.4
19
8.0
7.9
22
13
94.9
21
66.8
149
3.2
1.7
1.4
3.3
2.2
23
5.8
32
30
18
30
59
50
47
54
54
2.1
2.6
26
21
6.9
1.2
2.5
4.5
3.6
2.5
10
6.8
40
1
,4-dioxane
42
56
49
39
76
52
87
73
67
62
t-butyl methyl ether
Diethyl ether
Dichloromethane
Benzene
Heptane
Toluene
Cyclohexane
Hexane
Isooctane
8.8
33
74
86
66
80
73
122
2.9
1.8
2.7
13
12
9.0
14
13
87
85
a
Conditions: (RS)-11 (20 mM) in organic solvent (1 mL) was treated with vinyl acetate (5.40 mmol) in the presence of lipase (15 mg/mL).
b
Conversions were calculated from the enantiomeric excess (ee) of (R)-11 (substrate S) and (S)-10 (product P) for CAL CLEA and enantiomeric excess (ee) of
S)-11 ( substrate S) and (R)-10 (product P) for CRL using the formula: Conversion (C) = ee /(ee + ee ).
(
S
S
P
c
Enantiomeric excess of (R)-11 ( substrate S) for CAL CLEA and (S)-11( substrate S) for CRL 62316 determined by HPLC analysis (Daicel Chiralcel OJ-H column)
8
0:20; hexane/2-propanol, 1 mL/min flow rate at 254 nm.
Enantiomeric excess of (R)-10 (product P) for CAL CLEA and (S)-10 (product P) for CRL determined by HPLC analysis (Daicel Chiralcel OJ-H column) 80:20;
d
hexane/2-propanol, 1 mL/min flow rate at 254 nm.
e
)].28,41
E values were calculated using the formula: E = ln [(ee (1 – ee )/(ee + ee )]/ln[(ee (1 + ees)/(ee + ee
p s p s p P s
Chirality DOI 10.1002/chir

3
88
BANOTH ET AL.
Fig. 3. Effect of temperature on the CAL CLEA catalyzed
transesterification of (RS)-11 with vinyl acetate in 1,4-dioxane.
Fig. 2. Course of CRL 62316 catalyzed transesterification of (RS)-11 in
toluene.
various acyl donors (Table 3). Out of the six acyl donors
screened, vinyl acetate showed the best results; whereas
poor results were obtained with other acyl donors. Vinyl
acetate has the tautomerization ability of the in situ
generated vinyl alcohol to acetaldehyde. It shifts the equi-
librium to the product without acting as a competitive
substrate.
Effect of Temperature
The activity and enantioselectivity of the CAL CLEA and
CRL 62316 catalyzed kinetic resolution of (RS)-11 using vinyl
acetate as the acyl donor in 1,4-dioxane and toluene, respec-
tively, was determined at various temperatures. The conver-
sion and the enantiomeric excess were determined after the
resolution at 20, 25, 30, and 37 °C in 1,4-dioxane (Fig. 3)
and in toluene (Fig. 4), respectively. It was found that the con-
version (50%), the enantiomeric excess of the product (99.6%)
and substrate (98%), and enantiomeric ratio (E, 2641) were
highest in 1,4-dioxane at 37 °C (Fig. 3), while in the case of
toluene, the maximum conversion (49%), enantiomeric
excess of the product (98%) and substrate (95%), and enantio-
meric ratio (E, 447) were observed at 30 °C (Fig. 4).
Fig. 4. Effect of temperature on the CRL catalyzed transesterification of
(RS)-11 with vinyl acetate in toluene.
90 mg/mL) in 1,4-dioxane and toluene, respectively. These
experiments were conducted to understand the effect of
enzyme concentration on the conversion, enantiomeric excess,
and enantiomeric ratio of the product. In 1,4-dioxane, the
maximum enantiomeric ratio (3606), enantiomeric excess of
the product (99.7) and substrate (99) with 50% conversion
was achieved with 15 mg/mL CAL CLEA enzyme prepara-
tion (Fig. 5). In toluene, the conversion increased with the
increase in enzyme concentration up to a certain level, after
which the enzyme became nonselective. The maximum
Effect of Enzyme Concentration
Resolution was carried out using diverse concentrations of
CAL CLEA and CRL 62316 preparations (15, 30, 45, 60, and
a
TABLE 3. Effect of acyl donors on the transesterification of (RS)-11
a
a
CAL CLEA catalyzed in 1,4-dioxane
CRL 62316 catalyzed in toluene
b
c
d
e
b
c
d
e
Acyl donors
C (%)
ee
s
(%)
ee
p
(%)
E
C (%)
ee
s
(%)
ee
p
(%)
E
Acetic anhydride
Benzyl acetate
Ethyl acetate
Isopropenyl acetate
Phenylethyl acetate
Vinyl acetate
0.3
4.6
2.9
33
3.5
0.0
3.8
1.8
43
1.7
99
6.6
78
61
89
47
99
1.1
8.5
4.3
4.7
—
—
27
—
0.47
9.4
—
—
72
—
1.2
—
—
7.8
—
99
0.44
27
0.82
89
27
2.8
1057
49.9
50
90
55
a
Conditions: (RS)-11 (20 mM) in 1 mL solvent was treated with the acyl donor (5.4 mmol) at 30 °C in the presence of the lipase preparations (15 mg/mL).
b
c
S S P
Conversions were calculated from the enantiomeric excess (ee) of (R)-11 (substrate S) and (S)-10 (product P) using the formula: Conversion (C) = ee /(ee + ee ).
Enantiomeric excess of (S)-10 determined by HPLC analysis (Daicel Chiralcel OJ-H column) 80:20; hexane/2-propanol, 1 mL/min flow rate at 254 nm.
d
Enantiomeric excess of (R)-11 determined by HPLC analysis (Daicel Chiralcel OJ-H column) 80:20; hexane/2-propanol, 1 mL/min flow rate at 254 nm
e
28,41
E values were calculated using the formula: E = ln [(ee
p s p s p P s
(1 – ee )/(ee + ee )]/ln[(ee (1 + ees)/(ee + ee )].
Chirality DOI 10.1002/chir

SYNTHESIS OF ENANTIOPURE ACEBUTOLOL
389
Fig. 5. Effect of enzyme concentration on CAL CLEA catalyzed
transesterification of (RS)-11 in 1,4-dioxane.
Fig. 7. Effect of substrate concentration on CAL CLEA catalyzed
transesterification of (RS)-11 in 1,4-dioxane.
Fig. 6. Effect of enzyme concentration on CRL 62316 catalyzed
transesterification of (RS)-11 in toluene.
Fig. 8. Effect of substrate concentration on CRL catalyzed
transesterification of (RS)-11 in toluene.
enantiomeric ratio (214), enantiomeric excess of the product
(
97) and substrate (93) with a conversion of 49% was
Synthesis of (R) and (S)-Acebutolol (1)
obtained with 30 mg/mL CRL 62316 enzyme preparation in
toluene reaction medium (Fig. 6). For all the subsequent
experiments, an enzyme concentration of 15 and 30 mg/mL
of CAL CLEA and CRL in 1,4-dioxane and toluene were
used, respectively. Enantiomeric ratio (E) was calculated
from the values of ee and conversion using the equation as
described in footnote of Table 3.
From the enzymatic kinetic resolution of (RS)-11 with vinyl
acetate and CAL CLEA lipase enantiopure (R)-11 and (S)-10
was obtained. Optimum conditions for this reaction were:
organic solvent: 1,4-dioxane; time: 30 h; temperature: 37 °C;
enzyme concentration: 15 mg/mL and 20 mM substrate to
p
achieve C = 49.59%, E = 11325, ee = 99.91% and ee = 98.30%.
p
s
(
S)-10 was further hydrolyzed with K CO to afford (S)-11.
2 3
Effect of Substrate Concentration
Enantiopure compound (R)-11 was further reacted with
isopropylamine in MeOH in the presence of Et N under re-
It is necessary to study the effect of substrate concentration
on the activity and enantiomeric excess of any enzyme-
catalyzed chiral reaction. Various substrate concentrations
3
flux condition to afford (R)-acebutolol 1 (Yield 93%,
Scheme 8). Similarly, from the enzymatic kinetic resolution
of (RS)-11 with vinyl acetate and CRL 62316, lipase
enantiopure (S)-11 and (R)-10 was obtained. Optimum con-
ditions for this reaction were; organic solvent: toluene; time:
30 h; temperature: 30 °C; enzyme concentration: 30 mg/mL
and 10 mM substrate to achieve C = 49.59%, E = 239,
(
10, 20, 30, 40, and 50 mM) were used in the reaction mixture
for the resolution of (RS)-11 with CAL CLEA and CRL 62316.
In the case of CAL CLEA, it was found that maximum conver-
sion was obtained with 20 mM substrate (C = 49%, ee = 100%
and ee = 98%) (Fig. 7). In case of CRL 62316, it was found that
maximum conversion was obtained with 10 mM substrate
p
s
ee = 96.8%, and ee = 95.3%. Enantiopure compound (S)-11
p s
(
C = 49%, ee = 99% and ee = 95%) (Fig. 8).
was further reacted with isopropylamine in MeOH in pres-
p
s
ence of Et N under reflux condition to afford (S)- acebutolol
3
Deacylation of (RS) /(R)/(S)-10
1
(yield 90%, Scheme 8). Additionally, (R) and (S)-11 was
In aqueous K CO at room temperature, enantiopure, and
chemically synthesized by the reaction of epichlorohydrin
derivative (S) and (R)-2, respectively, with acetyl chloride
as a standard compound.
2
3
racemic halohydrin 11 was obtained from deacylation of 10
after 2 h reaction (Scheme 7).
Chirality DOI 10.1002/chir

3
90
BANOTH ET AL.
Scheme 7. Synthesis of (RS) /(R)/(S)-11.
Scheme 8. Chemo-enzymatic synthesis of (R) and (S)-acebutolol (1).
CONCLUSIONS
Efficient chemical and chemoenzymatic synthesis of the
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